The gram-negative bacterium, Actinobacillus actinomycetemcomitans (Aa), has been implicated in the etiology of localized juvenile periodontitis and other forms of periodontal disease. In addition, Aa has been responsible for endocarditis, as well as infections of the brain, thyroid, lungs, bone, and urinary tract. Aa has been reported to express several potential virulence factors, including leukotoxin, collagenase, cytotoxins, IgA protease, and endotoxin. With the exception of leukotoxin, whose gene has been cloned and sequenced, very little is known about the genes responsible for the production of virulence factors or their regulation. To understand the genetic basis of Aa pathogenesis will require the development of sophisticated tools for genetic analysis in this organism. The overall objectives of this proposal are (1) to gain experience in genetic studies with Aa, and (2) to develop effective methods and reagents for genetic manipulation of the Aa chromosome. Specifically, we intend to develop approaches for the construction of genetically marked deletion mutants of Aa, using the well characterized leukotoxin gene (IktA) as a model system. Two basic approaches will be explored (1) allelic exchange by cointegrate formation and resolution, and (2) directed deletion formation using the vector-mediated excision (VEX) system recently developed in this laboratory for genetic analysis of E. coli. In addition, homology-based recombination will be used to construct fusions of E. coli lacz gene to the Aa leukotoxin gene. It is anticipated that these studies will establish new methods for mutagenesis and manipulation of the Aa chromosome and produce new strains for evaluating the role of leukotoxin in Aa virulence.